Human Mammary Tumor Cell Line Research Articles

Evaluation of new migrastatin and dorrigocin congeners unveils cell migration inhibitors with dramatically improved potency

Lactimidomycin (LTM, 1), iso-migrastatin (iso-MGS, 2) and migrastatin (MGS, 3) are macrolide antitumor antibiotics differing in macrolide ring size but all bearing a glutarimide side chain. To further develop these natural products and related analogs as drug candidates we have produced and evaluated the biological activities of a small library of iso-MGS and LTM-derived agents; congeners evaluated bear either the MGS scaffold or related acyclic (dorrigocin) scaffolds. Scratch wound-healing (SWH) assays with 4T1 mouse and MDA-MB-231 human mammary tumor cell lines, respectively, reveal structural elements crucial to inhibition of cell migration by these compounds. Moreover, two substances, 14 and 17, with activity far superior to that of MGS are unveiled by SWH assays.

MMP-7 is involved in the aging of primary human mammary epithelial cells (HMEC)

Primary cultures of human mammary epithelial cells underwent significant morphological and functional changes during the aging process between passage 12 (P12) and passage 16 (P16). Concomitant with a progressive and significant expression of senescence-associated β-galactosidase as aging marker, the cells restructured their attachment, increased in size and ceased to divide. Young HMEC until P11 demonstrated a nearly 100% expression of distinct adhesion molecules such as CD24, integrin β1 (CD29) and CD44 similar to the human mammary tumor cell line MCF-7. In parallel with the aging-associated alterations of the cell adhesion, expression of CD24 and CD44 dropped in senescent P16 HMECs. However, levels of CD29 remained unchanged during the aging process. The tumor-associated Muc-1 (CD227), which was expressed to about 100% in the tumorigenic MCF-7 cells, was detectable in 51% of young HMEC in P11 and declined to 37% in aged HMEC in P16. In association with the remodeling of cell shape, expression levels of distinct matrix metalloproteinases including MMP-7 markedly decreased in aging HMEC. In contrast, MMP-1, MMP-2 and MMP-9 remained unchanged indicating a possible functional role of MMP-7 during the HMEC aging process. Indeed, down-modulation of MMP-7 by RNAi revealed a significantly elevated G 2/M cell cycle arrest and a 2- to 3-fold enhanced senescence-associated β-galactosidase expression as compared to control siRNA transfectants and control HMEC, respectively. Together, these findings suggested that decreasing MMP-7 expression contributes to accelerated aging of human mammary epithelial cells.

Susceptibility of cancer cells to herpes simplex virus-dependent apoptosis

Apoptosis has recently been associated with herpes simplex virus 1 (HSV-1) latency and disease severity. There is an intricate balance between pro- and anti-apoptotic processes during HSV-1 infection. When anti-apoptotic pathways are suppressed, this balance is upset and the cells die by apoptosis, referred to here as HSV-1-dependent apoptosis (HDAP). It has been observed previously that HeLa cancer cells exhibit an enhanced sensitivity to HDAP. Here, a series of specific patient-derived cancer cells was utilized to investigate the cell-type specificity of HDAP. The results showed that a human mammary tumour cell line was sensitive to HDAP, whilst syngeneic normal cells were resistant. Furthermore, low-passage-number primary human mammary epithelial cells were resistant to HDAP. When the susceptibility of human colon, brain, breast and cervical cancer cells was assessed, the only cells insensitive to HDAP were those resistant to all environmental stimuli tested. This implies that the HDAP resistance was probably due to mutations in the cellular apoptotic machinery. Thus, the susceptibility of cancer cells to HDAP requires that they possess a functional ability to undergo programmed cell death.

Hyaluronidase Expression Induces Prostate Tumor Metastasis in an Orthotopic Mouse Model

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.

Genistein, a soy isoflavone, up‐regulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NFκB

We have previously reported that estrogens up-regulate longevity-associated genes. As recent evidence has shown that estrogen replacement therapy is associated with an increased risk of cardiovascular disease, we have studied the effects of genistein, a soy isoflavone with a similar structure to estradiol, on the expression of antioxidant, longevity-related genes. MCF-7 cells (human mammary gland tumor cell line) were incubated for 48 h with 0.5 microM genistein, a concentration found in the plasma of populations consuming diets rich in soy protein. Peroxide levels were determined by fluorimetry, activation of extracellular-signal regulated kinase (ERK1/2), and nuclear factor kappaB (NFkappaB)-signaling pathways by Western blot analysis and ELISA, respectively, and mRNA expression of antioxidant genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Inhibition of basal peroxide levels in MCF-7 cells by genistein was prevented by pretreatment of cells with the estrogen receptor antagonist tamoxifen. Phosphorylation of extracellular regulated kinase (ERK)1/2 led to an activation of NFkappaB, as indicated by increased p50 subunit expression in nuclear extracts, and increased mRNA levels of the antioxidant enzyme manganese-superoxide dismutase (MnSOD). Inhibition of ERK1/2 abrogated genistein-mediated NFkappaB activation and elevated expression of MnSOD. Our molecular studies may provide a basis to determine the effects of genistein and other soy protein-derived products on longevity in both animals and the human population.

Identification of Human STAT5-dependent Gene Regulatory Elements Based on Interspecies Homology

STAT5 is a transcription factor essential for hematopoietic physiology. STAT5 functions to transduce signals from cytokines to the nucleus where it regulates gene expression. Although several important transcriptional targets of STAT5 are known, most remain unidentified. To identify novel STAT5 targets, we searched chromosomes 21 and 22 for clusters of STAT5 binding sites contained within regions of interspecies homology. We identified four such regions, including one with tandem STAT5 binding sites in the first intron of the NCAM2 gene. Unlike known STAT5 binding sites, this site is found within a very large intron and resides approximately 200 kb from the first coding exon of NCAM2. We demonstrate that this region confers STAT5-dependent transcriptional activity. We show that STAT5 binds in vivo to the NCAM2 intron in the NKL natural killer cell line and that this binding is induced by cytokines that activate STAT5. Neither STAT1 nor STAT3 bind to this region, despite sharing a consensus binding sequence with STAT5. Activation of STAT4 and STAT5 causes the accumulation of both of these STATs to the NCAM2 regulatory region. Therefore, using an informatics based approach to identify STAT5 targets, we have identified NCAM2 as both a STAT4- and STAT5-regulated gene, and we show that its expression is regulated by cytokines essential for natural killer cell survival and differentiation. This strategy may be an effective way to identify functional binding regions for transcription factors with known cognate binding sites anywhere in the genome.

Phosphorylation of ErbB4 on Tyrosine 1056 Is Critical for ErbB4 Coupling to Inhibition of Colony Formation by Human Mammary Cell Lines

In many studies, ErbB4 expression in breast tumor samples correlates with a favorable patient prognosis. Similarly, ErbB4 signaling is coupled to cellular differentiation and growth arrest in a variety of model systems. However, in some studies, ErbB4 expression in breast tumor samples correlates with poor outcome. Likewise, studies using some human mammary tumor cell lines suggest that ErbB4 is coupled to malignant phenotypes. Thus, the roles that ErbB4 plays in human breast cancer are still poorly defined. Here we demonstrate that a constitutively active ErbB4 mutant (ErbB4-Q646C) inhibits colony formation on plastic by two human mammary tumor cell lines (SKBR3 and MCF7) and by the MCF10A immortalized human mammary cell line, but does not inhibit colony formation by the MDA-MB-453 and T47D human mammary tumor cell lines. ErbB4 kinase activity is necessary for ErbB4 function and phosphorylation of ErbB4 Tyr1056 is necessary and appears to be sufficient for ErbB4 function. The inhibition of colony formation by MCF10A cells is accompanied by growth arrest but not cell death. These data suggest that ErbB4 behaves as a mammary tumor suppressor and that loss of ErbB4 coupling to growth arrest may be an important event in mammary tumorigenesis.

Growth of a human mammary tumor cell line is blocked by galangin, a naturally occurring bioflavonoid, and is accompanied by down-regulation of cyclins D3, E, and A

IntroductionThis study was designed to determine if and how a non-toxic, naturally occurring bioflavonoid, galangin, affects proliferation of human mammary tumor cells. Our previous studies demonstrated that, in other cell types, galangin is a potent inhibitor of the aryl hydrocarbon receptor (AhR), an environmental carcinogen-responsive transcription factor implicated in mammary tumor initiation and growth control. Because some current breast cancer therapeutics are ineffective in estrogen receptor (ER) negative tumors and since the AhR may be involved in breast cancer proliferation, the effects of galangin on the proliferation of an ER-, AhRhigh line, Hs578T, were studied.MethodsAhR expression and function in the presence or absence of galangin, a second AhR inhibitor, α-naphthoflavone (α-NF), an AhR agonist, indole-3-carbinol, and a transfected AhR repressor-encoding plasmid (FhAhRR) were studied in Hs578T cells by western blotting for nuclear (for instance, constitutively activated) AhR and by transfection of an AhR-driven reporter construct, pGudLuc. The effects of these agents on cell proliferation were studied by 3H-thymidine incorporation and by flow cytometry. The effects on cyclins implicated in mammary tumorigenesis were evaluated by western blotting.ResultsHs578T cells were shown to express high levels of constitutively active AhR. Constitutive and environmental chemical-induced AhR activity was profoundly suppressed by galangin as was cell proliferation. However, the failure of α-NF or FhAhRR transfection to block proliferation indicated that galangin-mediated AhR inhibition was either insufficient or unrelated to its ability to significantly block cell proliferation at therapeutically relevant doses (IC50 = 11 μM). Galangin inhibited transition of cells from the G0/G1 to the S phases of cell growth, likely through the nearly total elimination of cyclin D3. Expression of cyclins A and E was also suppressed.ConclusionGalangin is a strong inhibitor of Hs578T cell proliferation that likely mediates this effect through a relatively unique mechanism, suppression of cyclin D3, and not through the AhR. The results suggest that this non-toxic bioflavonoid may be useful as a chemotherapeutic, particularly in combination with agents that target other components of the tumor cell cycle and in situations where estrogen receptor-specific therapeutics are ineffective.

Rapamycin Disrupts Cyclin/Cyclin-Dependent Kinase/p21/Proliferating Cell Nuclear Antigen Complexes and Cyclin D1 Reverses Rapamycin Action by Stabilizing These Complexes

Rapamycin and its derivatives are promising anticancer agents, but the exact mechanisms by which these drugs induce cell cycle arrest and inhibit tumor growth are unknown. A biochemical analysis of human mammary tumor cell lines indicated that rapamycin-induced antiproliferative effects correlated with down-regulation of cellular p21 levels and the levels of p21 in cyclin-dependent kinase (Cdk) 2 and 4 complexes. Cyclin D1 overexpression reversed rapamycin action and this reversal correlated with increased levels of cellular p21, higher levels of p21 associated with Cdk2, and stabilization of cyclin D1/Cdk2/p21/proliferating cell nuclear antigen (PCNA) complexes. Experiments using a novel cyclin D1-Cdk2 fusion protein or a kinase-dead mutant of the fusion protein indicated that reversal of rapamycin action required not only the formation of complexes with p21 and PCNA but also complex-associated kinase activity. Similar results were observed in vivo. The rapamycin derivative RAD001 (everolimus) inhibited the growth of mouse mammary tumors, which correlated with the disruption of cyclin D1/Cdk2 complexes. The potential implications of these results with respect to the use of rapamycin derivatives in breast cancer therapy are discussed.

Tamoxifen resistance and adjuvant hormone therapy

The Oxford Overview of adjuvant endocrine trials [1] indicates that 5 years of adjuvant tamoxifen reduces recurrence by 41% and deaths by 34% in women with oestrogen receptor (ER)-positive breast cancers. At 5 years, in all patients studied, the recurrence rate was 25.8% in controls but 13.9% on tamoxifen. There was a substantial 'carry over' effect of tamoxifen such that even after 15 years of follow up mortality was about 30% less in tamoxifen-treated patients. The effect of tamoxifen was greater in patients with ER-positive, PR-positive as compared with ER-positive PR-negative tumours. These data indicate a substantial effect of tamoxifen but it is clear that approximately half of patients are resistant to tamoxifen de novo (early relapses) or acquire resistance if we assume that women who relapsed later had an initial response to tamoxifen. The potential reasons for resistance include activated growth factor pathways overriding the inhibitory effects of the drug either via nuclear or membrane ER. Of ER-positive PR-negative tumours, 30% are HER1/2-positive, as compared with about 10% of ER-positive PR-positive tumours, and this difference may account for their lower activity of tamoxifen in PR-negative tumours. Modern aromatase inhibitors (AIs) are more effective in reducing relapse compared with tamoxifen whether AI treatment is initiated after surgery (ATAC and BIG1-98 trials) or after 2–3 years of tamoxifen (ITA, ARNO/ABCSG). At present, it is difficult to distinguish any differences in effectiveness between the three agents used in these trials (anastrozole, letrozole and exemestane) but small differences in toxicity patterns are beginning to emerge. The reason for the greater effectiveness of AIs is not clear. In randomized studies of neoadjuvant endocrine therapy [2] and in the anastrozole adjuvant trials (ATAC and ARNO/ABCSG) [3], the AIs used were particularly more active than tamoxifen in the ER-positive PR-negative subgroup of tumours, but this was not seen in the BIG1-98 and IES trials. Studies on letrozole resistant human mammary tumour cell lines show that growth factor pathways such as MAPK (mitogen-activated protein kinase) are activated, and sensitivity to the AI can be restored by growth factor pathway inhibitors [4]. Also, AI resistance can be reduced by combined treatment with fulvestrant in animal models. These data suggest mechanisms whereby AI resistance may be circumvented in patients and point to new approaches to adjuvant treatment.

Additive estrogenic effects of mixtures of frequently used UV filters on pS2-gene transcription in MCF-7 cells

In order to protect consumers from ultraviolet (UV) radiation and enhance light stability of the product, three to eight UV filters are usually added to consumer sunscreen products. High lipophilicity of the UV filters has been shown to cause bioaccumulation in fish and humans, leading to environmental levels of UV filters that are similar to those of PCBs and DDT. In this paper, estrogen-regulated pS2 gene transcription in the human mammary tumor cell line MCF-7 was used as a measure of estrogenicity of four individual UV filters. Since humans are exposed to more than one UV filter at a time, an equipotent binary mixture of 2-hydroxy-4-methoxy-benzophenone (BP-3) and its metabolite 2,4-dihydroxy benzophenone (BP-1), as well as an equipotent multi-component mixture of BP-1, BP-3, octyl methoxy cinnamate (OMC) and 3-(4-methylbenzylidene) camphor (4-MBC), were also evaluated for their ability to induce pS2 gene transcription in order to examine additivity. An estrogen receptor-mediated mechanism of action was expected for all UV filters. Therefore, our null-hypothesis was that combined estrogenic responses, measured as increased pS2 gene transcription in MCF-7 cells after exposure to mixtures of UV filters, are additive, according to a concentration-addition model. Not all UV filters produced a full concentration–response curve within the concentration range tested (100 nM–1 μM). Therefore, instead of using EC 50 values for comparison, the concentration at which each compound caused a 50% increase of basal pS2 gene transcription was defined as the C50 value for that compound and used to calculate relative potencies. For comparison, the EC 50 value of a compound is the concentration at which the compound elicits an effect that is 50% of its maximal effect. Individual UV filters increased pS2 gene transcription concentration-dependently with C50 values of 0.12 μM, 0.5 μM, 1.9 μM, and 1.0 μM for BP-1, BP-3, 4-MBC and OMC, respectively. Estradiol (E2) had a C50 value of 4.8 pM. Experiments with equipotent mixtures all supported our null hypothesis that mixtures of UV filters act additively to activate the estrogen receptor (ER). In view of our results and observed plasma levels it cannot be excluded that daily exposure to sunscreen formulations may have estrogenic effects in humans.

Low-Dose Dietary Phytoestrogen Abrogates Tamoxifen-Associated Mammary Tumor Prevention

Wild-type erbB-2/neu transgenic mice were used to study the interactions between tamoxifen and dietary phytoestrogens (or isoflavones) by dose and form in vivo. Mice were randomized to one of four dietary formulas and implanted with an 8-week continuous-release tamoxifen or placebo pellet at 8 weeks of age. In placebo-treated mice, soy meal diet (but not diets supplemented with low-dose or high-dose isoflavones or a casein diet) resulted in prolongation of tumor latency. In tamoxifen-treated mice fed the soy meal, casein, or high-dose isoflavone enriched diets, the majority (>80%) showed no tumor formation by 60 weeks of age. Of the mice that developed tumors, latency was significantly prolonged. In tamoxifen-treated mice fed the low-dose isoflavone enriched diet, a much higher rate of mammary tumor development (>50%; P < 0.002) and a shorter tumor latency were observed. In vitro studies of human and mouse mammary tumor cell lines confirm that low doses of genistein, co-administered with tamoxifen, promote cell proliferation. This is in contrast to tamoxifen alone or tamoxifen with higher doses of genistein that are growth inhibitory. In summary, low-dose dietary isoflavones abrogated tamoxifen-associated mammary tumor prevention in vivo. These interactions are supported by in vitro data from human and mouse mammary tumor cell lines. These dose-associated interactions likely have relevance to the human use of tamoxifen for prevention or treatment of breast cancer.

GW9662, a potent antagonist of PPARγ, inhibits growth of breast tumour cells and promotes the anticancer effects of the PPARγ agonist rosiglitazone, independently of PPARγ activation

Peroxisome proliferator‐activated receptor gamma (PPARγ), a member of the nuclear receptor superfamily, is activated by several compounds, including the thiazolidinediones. In addition to being a therapeutic target for obesity, hypolipidaemia and diabetes, perturbation of PPARγ signalling is now believed to be a strategy for treatment of several cancers, including breast. Although differential expression of PPARγ is observed in tumours compared to normal tissues and PPARγ agonists have been shown to inhibit tumour cell growth and survival, the interdependence of these observations is unclear. This study demonstrated that the potent, irreversible and selective PPARγ antagonist GW9662 prevented activation of PPARγ and inhibited growth of human mammary tumour cell lines. Controversially, GW9662 prevented rosiglitazone‐mediated PPARγ activation, but enhanced rather than reversed rosiglitazone‐induced growth inhibition. As such, these data support the existence of PPARγ‐independent pathways and question the central belief that PPARγ ligands mediate their anticancer effects via activation of PPARγ.British Journal of Pharmacology (2004) 143, 933–937. doi:10.1038/sj.bjp.0705973

Tamoxifen induces apoptosis in Fas+ tumor cells by upregulating the expression of Fas ligand.

Tamoxifen (TAM), a nonsteroidal anticancer agent, is used in the treatment of breast cancer. In the current study, we investigated whether TAM induces apoptosis in tumor cells by altering the expression of Fas and Fas ligand (FasL). Several tumor cell lines were used to test the ability of TAM to induce apoptosis, which was studied using the TUNEL assay. The effect of TAM on the expression of Fas and FasL was analyzed using a flow cytometer. TAM was found to suppress the growth of an estrogen receptor-positive human mammary tumor cell line (T-47D) by inducing apoptosis. Interestingly, TAM also induced apoptosis in an estrogen receptor-negative murine T cell lymphoma cell line, EL-4. The ability of TAM to induce apoptosis in T-47D and EL-4 tumor cells correlated with the increased expression of FasL but not Fas on the tumor cells. Similar to TAM, a metalloproteinase (MP) inhibitor, which is known to increase the expression of membrane-bound FasL, was found to induce apoptosis in both T-47D and EL-4 tumor cells by increasing the expression of FasL but not Fas. Furthermore, both TAM and the MP inhibitor failed to induce apoptosis in L1210 tumor cell lines that failed to express FasL. The current study demonstrates that TAM can induce apoptosis in Fas(+) tumor cells by upregulating FasL.

Effects of the Phytoestrogens Genistein and Daidzein on BRCA2 Tumor Suppressor Gene Expression in Breast Cell Lines

A high intake of isoflavones is associated with a reduction of breast cancer risk among Japanese women. The aim of this study was to quantify BRCA2 tumor suppressor gene expression after treatment of cells with the phytoestrogens daidzein and genistein, the main compounds of soy. The effects of 5μg/ml genistein and 20μg/ml daidzein on BRCA2 expression were studied in two human mammary tumor cell lines, MCF7 andMDA-MB-231, and one normal human breast epithelial cell line, MCF10a. BRCA2 mRNA was evaluated by quantitative real time RT-PCR and the amount of BRCA2 protein was measured by affinity chromatography. With genistein, we observed a 60% increase of BRCA2 mRNA in MDA-MB-231 and MCF10a, which are, respectively, estrogen receptors a-/β+and α-/β-, and no variation in MCF7, which is ERα+/β+. Daidzein had no effect on BRCA2 mRNA expression. The level of BRCA2 protein with both food components also remained unchanged in all three cell lines. This suggests regulation of BRCA2 between the mRNA and protein levels. Treatment with actinomycin D and cycloheximide demonstrated that the increase in BRCA2 mRNA was not blocked by cycloheximide, indicating that de novo protein synthesis was required in MDA-MB-23, although de novo protein synthesis was not required in MCF10a for the genistein. With actinomycin D, genistein had a positive action on the transcriptional level of BRCA2 mRNA in MDA-MB-231 and MCF10a.Theuse ofananti-estrogen suggested that the action of daidzein and genistein on BRCA2 expression in human breast cell lines might not be mediated through the ER.

Growth Factor-specific Signaling Pathway Stimulation and Gene Expression Mediated by ErbB Receptors

The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1beta acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1beta acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2beta acting through the same heterodimeric receptor species. Surprisingly, NRG1beta and NRG2beta also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes.

Acetylenehexacarbonyldicobalt complexes, a novel class of antitumor drugs

Acetylenehexacarbonyldicobalt complexes were synthesized and tested for antitumor activity. The MCF-7 and MDA-MB-231 mammary tumor cell lines and the LNCaP/FGC prostate carcinoma cell line were used as in vitro models. The structural evaluation was performed by IR and NMR spectroscopy and revealed a change of the linear acetylene core to a structure comparable to Z-olefins after coordination to the cobalt centers. In cell culture experiments the strongest effects were found for hexacarbonyl[2-propinylacetylsalicylate]dicobalt ( 10), which was more active than cisplatin on the human mammary tumor cell lines MCF-7 and MDA-MB-231 in each concentration tested (a 5 μM concentration of this compound even caused cytocidal effects). In contrast to this, 10 influenced the growth of the LNCaP/FGC cells only marginally, even in the highest concentration. The mode of action of the complexes tested is unknown. As the cobalt complexes show strong antiproliferative effects and their ligands do not it could be unambiguously demonstrated that complex formation is essential to achieve cytotoxic effects.

Identification of a YAC from 16q24 carrying a senescence gene for breast cancer cells.

We have identified a 360 kb YAC that carries a cell senescence gene, SEN16. In our earlier studies, we localized SEN16 within a genetic interval of 3 - 7 cM at 16q24.3. Six overlapping YACs spanning the chromosomal region of senescence activity, were assembled in a contig. Candidate YACs, identified by the markers located in the vicinity of SEN16, were retrofitted to introduce a neo selectable marker. Retrofitted YACs were first transferred into mouse A9 cells to generate A9/YAC hybrids. YAC DNA present in A9/YAC hybrids was further transferred by microcell fusion into immortal cell lines derived from human and rat mammary tumors. YAC d792t2 restored senescence in both human and rat mammary tumor cell lines, while an unrelated YAC from chromosome 6q had no senescence activity.

Molecular analysis of the inhibition of monocyte chemoattractant protein-1 gene expression by estrogens and xenoestrogens in MCF-7 cells.

Xenoestrogens (XEs) are a diverse group of chemicals that mimic estrogenic actions and may have adverse effects on human health. The influence of these compounds on cytokine production or immune system function remains unclear. In this study we have examined the effects of 17beta-estradiol (E2) and XEs on chemoattractant cytokine (chemokine) production and analyzed the molecular mechanism. Monocyte chemoattractant protein-1 (MCP-1), also termed monocyte chemotactic and activating factor, is a member of the chemokine family and attracts mainly blood monocytes. Human mammary tumor cell line MCF-7 cells produce a large quantity of MCP-1 in response to interleukin-1alpha (IL-1alpha). Addition of E2 to MCF-7 cells inhibited MCP-1 production in a dose-dependent manner. XEs, bisphenol A, and NP also inhibited MCP-1 production, although the potency was 3-4 orders of magnitude lower than that of E2. E2, bisphenol A, and NP inhibited MCP-1 messenger RNA expression in MCF-7 cells. Two closely located nuclear factor-kappaB sites, A1 and A2, have been identified in the promoter of the human MCP-1 gene. A luciferase construct containing this enhancer region (pGLM-ENH) was activated by IL-1alpha, and a mutation at either the A1 or A2 site resulted in a loss of IL-1alpha responsiveness. Treatment with E2 or XEs decreased the IL-1alpha-inducible pGLM-ENH luciferase activity significantly. In an electrophoretic mobility shift assay and supershift analysis, we found that treatment with E2 or XEs diminished the IL-1alpha-induced complex formation with both A1 and A2 probes, which was identified immunochemically to consist of nuclear factor-kappaB, p50, and p65. The IL-1alpha-induced p50/c-Rel complex to the A2 probe was also, to a lesser extent, decreased by E2 or XE treatment. The effects of E2 and XEs on the expression of MCP-1 seem to be much more dramatic than the effects of these agents on the promoters used in the luciferase assay, suggesting the involvement of an additional site(s) of the promoter region of the MCP-1 gene or posttranscriptional regulation of MCP-1 gene expression by E2 and XEs. This work represents the first report describing possible regulation of immune system function by XEs through inhibiting chemokine production.

Differential cross-talk of estrogen and growth factor receptors in two human mammary tumor cell lines

Cultured human mammary MCF7 and T47D tumor cell lines were used to test the interference of the partial antiestrogen 4′-hydroxytamoxifen (4-OH-TAM) and the pure antiestrogen ZM 182780 with growth factor (IGF-I, heregulin) signaling pathways. Growth of both cell lines was stimulated by IGF-I (20 ng/ml) or heregulin (3 nM). ZM 182780 effectively blocked growth factor induced as well as basal proliferation of MCF7 cells while the compound was ineffective in interfering with growth factor mitogenic activity in T47D cells. On both cell lines the IGF-I or heregulin- induced proliferation was enhanced further by 4-OH-TAM. This synergism could be inhibited dose-dependently by ZM 182780. When cells were grown in the presence of estradiol plus growth factors, the antiestrogenic potencies of both compounds and the efficacy of ZM 182780 were unaffected, while the efficacy of 4-OH-TAM was reduced. Our data show cell type specific cross-talk between the receptor for estrogen and that for IGF-I or heregulin, which is different in MCF7 and T47D cells, respectively. In MCF7 cells with demonstrable cross-talk, a clear superiority exists for a pure antiestrogen over a partial agonist in interfering with growth factor mitogenic activity.